Magnetic support of the optical emission line filaments in NGC 1275.

The giant elliptical galaxy NGC 1275, at the centre of the Perseus cluster, is surrounded by a well-known giant nebulosity of emission-line filaments, which are plausibly in excess of 10(8) years old. The filaments are dragged out from the centre of the galaxy by radio-emitting 'bubbles' rising buoyantly in the hot intracluster gas, before later falling back. They act as markers of the feedback process by which energy is transferred from the central massive black hole to the surrounding gas. The mechanism by which the filaments are stabilized against tidal shear and dissipation into the surrounding extremely hot (4 x 10(7) K) gas has been unclear. Here we report observations that resolve thread-like structures in the filaments. Some threads extend over 6 kpc, yet are only 70 pc wide. We conclude that magnetic fields in the threads, in pressure balance with the surrounding gas, stabilize the filaments, so allowing a large mass of cold gas to accumulate and delay star formation.

Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation.

The transferrin receptor (TfR) of reticulocytes is released in vesicular form (exosomes) during their maturation to erythrocytes. The heat shock cognate 70-kDa protein (Hsc70) has been demonstrated to interact with the cytosolic domain of the TfR and could thus trigger the receptor toward this secretion pathway. We investigated the characteristics of the interaction between Hsc70 and the TfR in exosomes with an in vitro binding assay using TfR immobilized on Sepharose beads and purified Hsc70. The results show that Hsc70 binds to exosomal TfR with characteristics expected of a chaperone/peptide interaction. We demonstrated that heat-denatured luciferase competed for in vitro binding, dependent on the nucleotide bound to Hsc70, and that this interaction activates the ATPase activity of Hsc70. Moreover, we used immunosuppressive agents that interact with Hsc70, thus decreasing Hsc70 binding to TfR in our in vitro binding assay and enabling us to assess the role of this interaction in vivo during reticulocyte maturation.

Mutated-gamma-actin restores growth to a yeast amino acid transport defective mutant.

A mutated yeast cell 22574d lacking all three proline transporters, PUT4, UGA4, and GAP1, and incapable of growth on proline recovers its lost ability to grow on proline as sole nitrogen source when transformed with a mutagenized mouse gamma-actin cDNA (M-gamma-A). Native mouse gamma-actin cDNA is ineffective. The 3'-region of gamma-actin cDNA was mutagenized to resemble E51 cDNA previously isolated from Ehrlich tumor cells. The E51 cDNA has an extended reading frame in the 3'-region compared to that in native gamma-actin. The extension of the open reading frame in E51 cDNA, was found to be due to an additional pair of bases (TG) at position 1104 of E51 cDNA. After site-directed mutagenesis of the 3'-region of native gamma-actin cDNA to resemble that of E51 cDNA, the construct, M-gamma-A cDNA, was expressed in the 22574d yeast. While the transformation with M-gamma-A increased the uptake of both proline and gamma-amino butyric acid, the transport of five other solutes was not changed by this transformation. Northern blotting of the nontransformed and the M-gamma-A-transformed 22574d cells with gene-specific probes for the three proline transporters showed the expression of an mRNA for UGA4 in both transformed and nontransformed cells but no evidence for the expression of GAP1 or PUT4. The mRNA for UGA4 was expressed at a lower level in strain 22574d than in the parent yeast sigma1278b. Furthermore, the message in the mutated cells is smaller in size by about 15%. These results are consistent with the synthesis of a mutated transporter which requires the coexpression of M-gamma-A, but not native gamma-actin, to restore physiological function, i.e., proline or gamma-amino acid transport.

Loss of glucose transport in developing avian red cells.

Although red cells are generally associated with significant glucose transport and dependence on glycolysis, the mature red cells of some species (e.g. pig) show very low glucose transport. The generally low level of glucose transport in mature mammalian red cells is the result of maturational development, since it has been shown that even in red cells which have negligible glucose transport (e.g. pig red cells) the corresponding reticulocytes have significant glucose transport activity. The reticulocytes of the chicken, however, show minimal glucose transport activity. But this also is the result of maturational development, since chicken bone marrow red cells do transport glucose which diminishes upon cell maturation in vitro. The erythroblast chicken cell line, HD3, has high glucose transport activity which is lost upon induction to the red cell phenotype. Growing HD3 cells have much higher levels of transport than native chicken bone marrow cells and this is associated in part with elevation of glucose transporter (GLUT) mRNAs as a consequence of the expression of the v-erbA and v-erbB oncogenes. Both native bone marrow red cells and HD3 cells, when incubated in vitro under conditions where maturation occurs, show substantial losses of GLUT mRNA and GLUT proteins. To assess whether the inducers of maturation (hemin and butyrate) affect only the normally expressed GLUTs, chicken GLUT3 expressed from a different promoter was introduced into the HD3 cell by retroviral infection. Both the endogenous and exogenous transporters were lost upon cell differentiation and maturation, leaving a cell with low glucose transport activity. Conversely, in growing cells, butyrate had a pronounced effect on the elevation of the GLUT3 mRNA, especially on the exogenous GLUT3 mRNA, and elevated glucose transport prior to differentiation. These results are consistent with the conclusion that chicken red cell development involves a requirement to reduce glucose transport activity. The near absence of glucose transport in the embryonic chicken red cell is thus due to a loss of this transporter during early development which occurs at an earlier developmental stage in the chicken red cell than in the mammalian red cell.

Expression cloning of a mammalian amino acid transporter or modifier by complementation of a yeast transport mutant.

A cDNA clone named L21 was isolated from L6 rat muscle cells by complementation of a yeast proline transport mutant. L21 cDNA has 2,268 bp and codes for a peptide of 228 amino acids with four potential transmembrane domains. The amino acid sequence of L21 shows no homology to any known proteins. Expression of L21 cDNA enables the mutant yeast to grow in proline as the sole nitrogen source and to transport proline, alpha-aminoisobutyric acid (AIB) and leucine. The Km for proline is about 1.0 mM. The substrate specificity of L21 expressed in yeast shows no striking similarity to known mammalian amino acid transporters.

Cleavage of the transferrin receptor by human granulocytes: differential proteolysis of the exosome-bound TFR.

In contrast with other mammalian granulocytes, human granulocytes rapidly cleave the transferrin receptor (TFR) from sheep exosomes. Proteolysis of TFR from exosomes is more rapid and more extensive than that from the sheep reticulocyte cell surface itself, although little difference in cleavage is seen when immunoprecipitates or when immobilized, solubilized receptors from the two sources are compared. Circulating exosomes but not the plasma membrane fraction from seven species of immature red cells or erythropoietic cells show the presence of a peptide of approximately 18 kD recognized by an antibody to the cytoplasmic domain of the TFR. This 18 kD peptide is virtually absent from the corresponding cellular plasma membranes including human reticulocyte membranes. Taken together, the data are consistent with the conclusion that the exosomes released to the circulation from maturing red cells are the principal source of the soluble, circulating, truncated TFR. The granulocyte protease appears to be present on the cell surface and not released into the medium after short (30 min) periods of incubation at 37 degrees C. The protease is inhibited by PMSF but only at high (1 mM) concentrations. Using sheep exosome bound-TFR as substrate, human granulocytes exceed other granulocytes in their capacity to cleave TFR, suggesting that this may be a key factor for the prominent amount of circulating, soluble receptor found in human sera during periods of elevated reticulocyte levels. Human exosomes, unlike those from other species, contain little native size TFR. Truncated receptor containing the cytoplasmic domain being predominant in human exosomes.

Is gamma-actin a regulator of amino acid transport?

A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells. The cloned cDNA (E51) was found to be 90% homologous to gamma-actin. Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5. The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei. A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA. Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells. The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth. The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport.

Long-term trends and sources of organochlorine contamination in Canadian tundra Peregrine Falcons, Falco peregrinus tundrius.

Levels of eggshell thinning, and organochlorine residues in egg contents, blood plasma of adults and juveniles, tissue samples, and prey species were determined for a population of migratory Peregrine Falcons (Falco peregrinus tundrius) breeding in the Canadian Arctic. Temporal trends were assessed by comparing data collected during 1991-1994, with data from 1982-1986, for the same population. Shells (n=54) from 1991-1994 averaged 15% thinner than eggs produced prior to the introduction of DDT. No improvement in shell thickness was detected between decades. Mean DDE residue levels in eggs showed a decline from 7.6 mg kg (1982-1986) to 4.5 mg kg (1991-1994), but there was no significant change in SigmaPCB residues. Moreover, the proportion of clutches with eggs exceeding critical SigmaPCB, DDE, and dieldrin residue levels (10%) did not change between decades. Relative to Greenland and Alaskan populations, F. p. tundrius at Rankin Inlet show high levels of organochlorine contamination and little reduction in residues over the last decade. These Tundra Peregrines continue to be exposed to organochlorines in Latin America; however, results also link relatively high levels in the study population with waterfowl species that do not leave Canada in winter.

Identification of the integrin alpha 3 beta 1 as a component of a partially purified A-system amino acid transporter from Ehrlich cell plasma membranes.

We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.

Hsp-70 is closely associated with the transferrin receptor in exosomes from maturing reticulocytes.

The presence of the heat shock protein (hsp-70) has been detected in exocytosed vesicles (which are named exosomes) from mammalian and avian immature red cells (i.e. reticulocytes) as well as from a differentiating avian erythroleukaemic cell line. The close, but non-covalent, association of hsp-70 with the transferrin receptor (TFR) in exosomes is demonstrated by: (1) the ability to cross-link hsp-70 to TFR; (2) the co-immunoprecipitation of hsp-70 and TFR with an antibody against TFR, and the co-immunoprecipitation of TFR and hsp-70 with antibody against hsp-70; and (3) the retention of TFR by hsp-70 bound to ATP-agarose and the simultaneous elution of both proteins by excess ATP. Semi-quantitative analysis of the relative efficiency of cross-linking of these proteins in exosomes versus plasma membranes shows that TFR in exosomes is preferentially bound to hsp-70. From an analysis of the relative amounts of hsp-70 and TFR regenerated from the cross-linked complex, the ratio of TFR monomer bound to hsp-70 is approximately 1.5 to 1. Given the presence of hsp-70 in exosomes from several species and its close association with TFR (the major protein lost during reticulocyte maturation) it is proposed that hsp-70 plays a role in exosome formation and/or release in immature red cells.

Differentiation of two classes of "A" system amino acid transporters.

Injection of mRNA from GF-14 cells (a mouse lymphocyte cell line) into Xenopus oocytes led to the expression of a transport activity characteristic of amino acid transport system A, i.e., Na+ dependent and recognizing aminoisobutyric acid and its methyl derivative (AIB and meAIB). Sucrose density gradient fractionation of GF-14 mRNA showed that the maximum level of expression of activity was associated with a 2.2-kb RNA fraction. Pretreatment of GF-14 cells with insulin caused a twofold increase in system A transport activity in the cells themselves and the mRNA from the insulin-treated cells induced a comparable increase in A system transport over control mRNA when expressed in oocytes. mRNA from cells treated with insulin in presence of actinomycin D did not show a response to insulin, suggesting that GF-14 cells synthesized new transporter in response to insulin. Similar experiments with mRNA from the Ehrlich ascites cells, showed that expression of system A type transport was associated chiefly with a 4.2-kb mRNA fraction. Although insulin treatment of Ehrlich cells also caused an increase in A system transport activity in the cells themselves (nearly twofold), the response to insulin was not blocked by actinomycin D or cycloheximide. Moreover, mRNA from insulin-treated and untreated Ehrlich cells gave the same level of expression in oocytes of A type amino acid transport. In contrast to GF-14 cells, Western blots of plasma membranes showed that insulin treatment of Ehrlich cells increased the amount therein of a 120- to 130-kDa peptide, previously associated with amino acid transport (Proc. Natl. Acad. Sci. USA 85, 7877, 1988), suggesting that in the Ehrlich cells, but not in GF-14, cells this polypeptide was translocated from intracellular sites in response to insulin. The data associate two distinctly different responses to insulin with A-type amino acid transporters and are consistent with the existence of more than a single A type amino acid transporter in mouse cells.

Loss of glucose transporters is an early event in differentiation of HD3 cells.

The HD3 cell, a chicken erythroblast cell line infected with a temperature-sensitive avian erythroblastosis virus, becomes committed to differentiate to an erythrocyte upon temperature shift in presence of inducers. Before induction, the HD3 cell transports glucose and 2-deoxyglucose (2-DG). 3-O-methylglucose is poorly taken up. Upon induction of differentiation, glucose and 2-DG transport activity fall. Twenty-four hours postinduction, up to 75% of the glucose transport activity may disappear. By use of cDNA probes for chicken glucose transporters, two species of mRNA of 3.1 and 1.7 kb (equivalent to mammalian GLUT1 and GLUT3 mRNA, respectively) are detected. Both messages virtually disappear within 48 h after induction. Run-on assays show the cessation of synthesis of the corresponding RNAs parallel to the loss of glucose transport. In contrast to the glucose transporters, the nucleoside transporter level increases after induction of hematopoiesis. This developmental pattern is consistent with earlier studies showing that mature chicken erythrocytes have little glucose transport activity but retain appreciable levels of the nucleoside transporter and that nucleosides and glutamine provide major sources of oxidizable carbon compounds to sustain metabolism in circulating chicken erythrocytes.

Nucleosides and glutamine are primary energy substrates for embryonic and adult chicken red cells.

It has been assumed that glucose is a major energy yielding substrate for chicken red blood cells. In this report we show that glucose fails to maintain cellular ATP levels in embryonic and mature chicken erythrocytes during overnight incubation. Of over a dozen metabolites tested, inosine, guanosine, and glutamine were the most efficacious ATP-sustaining substrates. Of seven potential citric acid cycle substrates, only glutamine significantly sustained ATP levels. Incubation with inosine plus glutamine sustained the ATP level at over 70% of the initial value found in embryonic chicken red cells. Uridine, cytidine, xanthosine, glutamate, and pyruvate were ineffective substrates. Similar results were obtained with adult erythrocytes, except that glutamine plus inosine fully sustained ATP levels during long-term incubation. Adenosine did not sustain ATP levels. Below 1 mM, the adenosine level was rapidly exhausted and above 1 mM its presence led to cell lysis. The ability of some nucleosides to maintain ATP levels is consistent with the high numbers of nitrobenzylthioinosine binding sites (nucleoside transporter sites) both in mature chicken red cells (approximately 10,000 sites/cell) and in embryonic red cells (approximately 30,000 sites/cell). Unlike pig red cells which switch from glucose to nucleosides at the erythrocyte stage, chicken cells show primary dependence on nucleoside metabolism at the embryonic stage.

Membrane properties of erythrocytes in subjects undergoing multiple blood donations with or without recombinant erythropoietin.

To examine the characteristics of 'young' human red cells, we studied blood from seven healthy male volunteers who developed systemic reticulocytosis during a 3-week blood donation period. Each of these subjects donated a total of 6 units (450 ml/unit) of blood (2 units/week for 3 weeks) with subcutaneous recombinant erythropoietin (SC rEPO; 200 U/kg daily for 3 weeks). Two of these subjects were also studied with a similar protocol in the absence of rEPO (4.5 +/- 0.5 units donated). SC rEPO administration was associated with an increased K content of the erythrocyte and with the appearance of hypochromic cells, which were initially normocytic and then became normochromic and microcytic. Measurements of cation transport revealed that, with the exception of the Na-K-Cl cotransport, all the systems studied increased their activities following blood donations with or without SC rEPO. The increase was highest in the K-Cl cotransport (2- and 5-fold for control and rEPO parts of the study, respectively), while the Na-K pump increased slightly in control and 40% with rEPO. The Na-Li countertransport increased 40% and 100% in the control and rEPO parts of the study, respectively. Concomitant with increased ion transport activity, electron microscopic studies of plasma and red cells of subjects receiving SC rEPO showed the presence of circulating exosomes and cytoplasmic multivesicular bodies. The transferrin receptor was detected in the circulating exosomes, thereby providing evidence that, as do nonhuman red cells, maturing human reticulocytes shed exosome-associated transferrin receptors.

Origin of a soluble truncated transferrin receptor.

It has recently become evident that elevation of reticulocytes in the circulation of several species, including humans, leads to the formation of a noncellular transferrin receptor (TFR). In humans, the majority of the released receptor is in truncated form (Shih et al: J Biol Chem 265:19077, 1990). In other species (sheep, rat, chicken) the receptor is associated with a vesicle (exosome) and is full length (Johnstone et al: J Cell Physiol 147:27, 1991). In this report we show that in sheep reticulocytes incubated in vitro, the majority (approximately 75%) of the released receptor is of native size and is exosome associated. A fraction (approximately 25%) is a truncated form of approximately 80 Kd corresponding to the exofacial domain of the TFR. Herein we also address the question of whether the truncated receptor originates by proteolytic cleavage directly from the cell surface or by cleavage from exosomes. Using surface 125I-labeled sheep reticulocytes as the experimental model, we show that during in vitro maturation, 125I-TFR of native size appears in exosomes before the soluble, truncated, exofacial domain of the receptor is detected in the medium. Because cleavage and release of the exofacial domain would likely leave the truncated cytoplasmic and transmembrane domains in the originating membrane (plasma membranes or exosomes), both fractions were probed with antibodies specifically generated against the cytoplasmic domain of the receptor. Only exosomes, not plasma membranes, show the presence of a approximately 17-Kd peptide recognized by the antibody to the cytoplasmic domain of the transferrin receptor. Thus, it is concluded that the truncated, soluble receptor originates from exosomes in sheep. A 17-Kd cytoplasmic domain of the TFR was also detected in exosomes from the reticulocytes of an anemic man, suggesting that the truncated receptor in man may also originate from exosomes. Using in vitro cultures of surface 125I-labeled sheep reticulocytes, it is concluded that exosome formation is the principal route for maturation-associated loss of the TFR. A similar conclusion was made earlier (Johnstone et al: J Cell Physiol 147:27, 1991) for the nucleoside transporter of maturing sheep reticulocytes.

Expression and loss of the transferrin receptor in growing and differentiating HD3 cells.

During induced differentiation and maturation of HD3 cells (a chicken erythroblast cell line infected with a temperature-sensitive mutant of the avian erythroblastosis virus), the levels of transferrin receptor (TFR) and nucleoside transporter increase. Both these activities increase before elevated levels of hemoglobin are detected. Shortly after induction, as cellular TFR levels rise, a native-size TFR is detected in the cell-free culture medium, associated with an exosome fraction (100,000 xg pellet). Nucleoside transporter (measured as NBMPR-binding activity) is not increased in this pellet with induction. Previous studies have suggested that exosome formation in peripheral reticulocytes may be a significant route for loss of specific membrane proteins (Johnstone et al., 1991). Although the present experiments in HD3 cells do not address the quantitative importance of exosome formation, the studies suggest that exosome formation is an early event in commitment to the red cell lineage and is not a phenomenon restricted to the terminal stages of red cell maturation.

Evidence for an internal pool of nucleoside transporters in mammalian reticulocytes.

Comparisons of the site specific binding of nitrobenzylthioinosine (NBMPR) to intact and lysed red cells from various mammalian and avian species suggest the presence of a cytoplasmic pool of nucleoside transporters. In some species the cytoplasmic pool is about 50% of the total (mouse). On the average, the cytoplasmic pool is approx. 20% of the surface pool of NBMPR-binding sites. In sheep reticulocytes, both pools disappear in an energy-dependent manner during the maturation of the reticulocyte in vitro.

The Jeanne Manery-Fisher Memorial Lecture 1991. Maturation of reticulocytes: formation of exosomes as a mechanism for shedding membrane proteins.

The transferrin receptor is a member of a group of reticulocyte surface proteins that disappear from the membranes of reticulocytes as the cells mature to the erythrocyte stage. The selective loss of membrane proteins appears to be preceded by the formation of multivesicular bodies (MVBs). At the reticulocyte stage, many species of mammalian red cells including man, and one nucleated avian species (chicken), contain these intracellular structures in both natural and induced anemias. Also characteristic of blood containing reticulocytes is the presence of circulating vesicles (exosomes), which contain proteins and lipids characteristic of the plasma membrane. These exosomes appear to arise from the contents of the MVBs, after the fusion of MVBs with the plasma membrane. The proteins in the exosomes are those frequently lost during red cell maturation (e.g., transferrin receptor). The major transmembrane proteins (such as the anion transporter) are fully retained into the mature red cell, indicating a highly selective mechanism of recognition of a specific group of proteins. The exosomes are largely devoid of soluble proteins and proteins associated with lysozomes or mitochondria. A speculative model is proposed which addresses the questions of the maturation-induced structural changes in a class of membrane proteins, their recognition and selective loss involving exosome formation, and the release of exosomes to the circulation.

Intracellular localization of newly synthesized transferrin receptors in the peripheral sheep reticulocyte.

In this paper, we provide evidence for an incompletely glycosylated transferrin receptor (TfR) which is not transported to the plasma membrane in the sheep reticulocyte. Cleveland peptide maps of the native (preexisting) TfR and [35S]methionine-labeled TfR were different. If the receptors were deglycosylated before mapping, the peptides were identical. There was preferential binding of the [35S]TfR to Con A-Sepharose, indicating the existence of a higher density of high mannose chains on the 35S-labeled TfR. Moreover, when total [3H]mannose-labeled glycopeptides from reticulocytes were separated on a column of Bio-Gel P6, the [3H]mannose was associated with endoglycosidase H-sensitive high mannose or hybrid oligosaccharides, but not with complex sugars. After Percoll density gradient centrifugation, the [35S]TfR peaked in a fraction which separated from the bulk of the native TfR. The transmembrane glycoproteins, Band 3 and mature glycophorins, are not synthesized in the sheep reticulocyte. It appears that the reticulocyte, at this stage of red cell development, has lost the vesicles and/or proteins which are required to transport proteins from the site of translation to the cell surface.

Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins.

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.